Tissue preservation

ABSTRACT

A process for making and using a tissue preservation gel. The gel is comprised of water, reticulated acrylic acid, water soluble short-chain paraben, isopropyl alcohol and either triethanolamine or propylene glycol. To use the gel, a tissue to be preserved is first bathed in hypertonic sodium chloride, then soaked in a wash in multiple increasing concentrations of isopropyl alcohol and then preserved indefinitely in the tissue preservation gel.

CROSS-REFERENCES TO RELATED APPLICATIONS

None.

STATEMENT REGARDING FEDERAL SPONSORED RESEARCH OR DEVELOPMENT

None.

NAMES OF PARTIES TO A JOINT RESEARCH AGREEMENT

None.

REFERENCE TO A “SEQUENCE LISTING”, A TABLE, OR A COMPUTER PROGRAMLISTING APPENDIX SUBMITTED ON COMPACT DISC ANDINCORPORATION-BY-REFERENCE OF THE MATERIAL ON THE COMPACT DISCLOSURE

None.

STATEMENT REGARDING PRIOR DISCLOSURES BY AN INVENTOR OR JOINT INVENTOR

None.

BACKGROUND OF THE INVENTION 1. Field of the Invention

The present invention relates to preservation of biological tissue, andmore particularly, to an improved formula and method of use of a viscousfluid to preserve plant and animal tissue indefinitely.

2. Description of Related Art Including Information Disclosed Under 37CFR 1.97 and 37 CFR 1.98

Several designs for tissue preservation formulas have been designed inthe past. None of them, however, is non-toxic, non-flammable,non-carcinogenic, biodegradable while retaining performance toindefinitely preserve biological tissue without significant degradationof the tissue for later visual micro-analysis.

Applicant believes that the closest references correspond tocommercially available formaldehyde based products. Each of these, bynature of having formaldehyde, is potentially dangerous to those usingthe products and affects the tissues being preserved negatively so thatvisual analysis cannot be confidently performed.

Other patents describing the closest subject matter provide for a numberof more or less complicated features that fail to solve the problem inan efficient and economical way. None of these patents suggest the novelfeatures of the present invention.

A brief abstract of the technical disclosure in the specification andtitle are provided as well for the purposes of complying with 37 CFR1.72 and are not intended to be used for interpreting or limiting thescope of the claims.

Without limiting the scope of the invention, a brief summary of some ofthe claimed embodiments of the invention is set forth below. Additionaldetails of the summarized embodiments of the invention and/or additionalembodiments of the invention may be found in the detailed description ofthe invention below.

BRIEF SUMMARY OF THE INVENTION

It is one of the main object of the present invention to provide aformulation directed to the preservation and conservation of biologicaltissue samples.

Another object of this invention is to provide a preservation solutionwith the viscosity and impedance calculated to allow the biologicalsamples to be analyzed by diagnostic imaging, such as by microscope, MRIor TAC.

It is another object of the present invention to increase the weight ofthe solution part and container complex.

Another object of the present invention is to provide a solution thatallows management of the specific weight of the of the part to besubmitted to phantomization.

Another object of the present invention is to provide a long-termpreservation and maintenance solution that is non-toxic, biodegradable,non-flammable, thermally stable and with a jelling or freezing point atapproximately 20° C.

It is yet another object of this invention to provide such a formula andmethod of use that is inexpensive to manufacture and maintain whileretaining its effectiveness.

Further objects of the invention will be brought out in the followingpart of the specification, wherein detailed description is for thepurpose of fully disclosing the invention without placing limitationsthereon.

These and other embodiments which characterize the invention are pointedout with particularity in the claims annexed hereto and forming a parthereof. However, for a better understanding of the invention, itsadvantages and objectives obtained by its use, reference can be made tothe drawings which form a further part hereof and the accompanyingdescriptive matter, in which there are illustrated and described variousembodiments of the invention.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWING

With the above and other related objects in view, the invention existsin the details of construction and combination of parts as will be morefully understood from the following description, when read inconjunction with the accompanying drawings in which:

FIG. 1 shows a chart of the advantages of the present formula over thatof the prior art.

DETAILED DESCRIPTION OF THE INVENTION

While this invention may be embodied in many different forms, there aredescribed in detail herein specific embodiments of the invention. Thisdescription is exemplary of the principles of the invention and is notintended to limit the invention to the particular embodimentsillustrated and described.

For the purpose of this disclosure, like reference numerals in thefigures shall refer to like features unless otherwise indicated or isobvious by context.

The subject formulation and method of use is sometimes referred to asthe device, the invention, the preservation solution, the formula, thegel, the liquid, the preparation or other similar terms. These terms maybe used interchangeably as context requires and from use the intentbecomes apparent. The masculine can sometimes refer to the feminine andneuter and vice versa. The plural may include the singular and singularthe plural as appropriate from a fair and reasonable interpretation inthe situation.

Generally, the formula of the solution is aimed at the preservation andconservation of biological tissues. It has a viscosity and impedancecalculated to allow the pieces to be analyzed by a traditionaldiagnostic imaging methods, such as microscope, medical ultrasound, TACor MRI.

Preservation generally refers to protecting and caring for the integrityof a tissue sample from the moment it is received and intended to besubjected to the present process. Conservation generally has to do withthe steps to which the sample is subjected so that it remains in goodcondition over time. And in this method these two characteristics aremaintained through time indefinitely.

The formulation comprises to primary phases in the preparation. First,is histological fixation. In this phase the tissue from the sample isimmersed in the fixative solution for a period of no less than 24 hours.Then the usual and known histological fixation process is continued toachieve the histological cuts where the fixative will be the presentpreservation gel. Then, the conservation phase of the anatomical piecescontinue.

In a preferred method to achieve the histological fixation of thetissue, there are four steps. First, is fixing. A universal fixator thathas been effective in testing is commercially available Formol™. Othercommercially available gelling agents may also be effective. From themoment of exeresis of the anatomic piece or organ, it is immersed in avolume of the preservative gel of a volume approximately 5 to 10 timesthe volume of the anatomic piece being fixed. The anatomic piece remainsin the preserving gel no less than 24 hours to complete the fixingprocess.

Gelification is fairly described as the process of transformation of asampled substance into a gelatine form. Because of this process thesampled substance, including their liquid components, become solid withthe help of a gelling agent.

The next phase is inclusion. Here, the tissue is subject to dehydrationgiven by alcohols of increasing percentage concentrations. The last bathwill be the preserving gel in a container capsule or grid. The inclusionprocess is performed so that the tissue to be studied achieves thenecessary rigidity an adequate dehydration so that the tissue sample maybe associated with the paraffin block.

Third, is the cut phase. Once the paraffin block is achieved the tissueis cut with a microtome. The cut is then mounted on the sheets.

The fourth step in the process is staining. In this step the tissue isready to be stained with commercially available stains such asEmatocillin™ or Eosin™. The selection of the stain may be based on thebasic or alkaline chemical affinity. It is through this process thesheet containing the tissue to be study is ready to be visualizedthrough traditional microscopy means. In general, histological stainingagents are used to provide contrast in tissue sections to aid inmicroscopic analysis.

Conservation Process of Anatomical Parts

The following is an example of a typical process for the conservation ofanatomical pieces for forensic or educational purposes. For eitherpurpose, the preservation of parts or whole organisms is done under theprocess of severe general dehydration of all the systems of which it iscomprised. Known substances are used which the main effect isdehydration and also agents that prevent the over-production of bacteriaand fungi to avoid the consequent putrefaction and fermentation of thetissue.

The present preservation formula is provided under the format ofconstant immersion of the biological part to be preserved. It isessential to avoid contact with common air and its subsequent oxidation.Together with this, it is intended with this immersion environment tocontrol factors that also affect the parts such as the maceration ordegradation of the internal environment has been widely demonstratedwith pieces exposed and stored in glass containers for a long time. Inthese processes the penetration of Formalin™ (or other formaldehyde basesolutions) are already known to penetrate about 0.5 mm per hour. It thenreaches its maximum expression, consuming and disintegrating organs andsystems of the biological pieces, making it not be optimal for futuredissections.

The following describes an example of the process for conservation ofanatomical samples comprised of two primary steps:

First is the washing and permeabilization phase. This is comprised ofimmersing the piece in about a 0.9% hypertonic sodium chloride solution.The volume guideline for amount of this solution is about that of thetissue piece multiplied by about ten times. This result gives theapproximate volume of the 0.9% hypertonic sodium chloride solution thatwill be prepared for the washing. In most cases, previous exsanguinationand lavage of excretory tracts has been completed.

The second phase is immersion in the presently disclosed tissuepreservation formula. Using the same sample volume calculation guidelineas for the permeabilization phase, the volume of the preservation fluidneeded is calculated. The tissue sample is immersed in and sealed in itscontainer with as little air as possible to reduce the potentialdegradation effects of oxygen and other contamination.

Example of the Preservation Fluid Formula and Process of Manufacture

1. 1000 mL Distilled water

2. 5 g Carbopol 940™ (a standardized polymer of acrylic acidcross-linked with a polyfunctional compound)

(a) Alternatively 5 g reticulated acrylic acid and it's derivatives

3. 1 mL triethanolamine (98-99% pure)

(a) Alternatively 1 mL propylene glycol USP/EP may be substituted

4. 650 mL Isopropyl alcohol (100%)

5. 10 mL glycerin

6. 1 g Nipagin M™ (a standardized water soluble short-chain paraben)

(a) Alternatively 1 g sodium benzoate may be substituted

7. Optionally, 0.5 mL blue dye

8. Optionally, 5 mL fragrance

To prepare the tissue preservation liquid: In a 500 cc cylinder, place350 cc of distilled water. Then, 5 g of Carbopol™ and 1 g of Nipagin™are added to the water. With a glass stirrer, mix vigorously. Then themouth of the cylinder is covered with control paper for twenty fourhours. It should be appreciated that Nipagin™ is a trade name for astandardized water soluble short-chain paraben, for example, amethylparaben. It should be appreciated that Carbopol™ is a trade namefor a standardized polymer of acrylic acid cross-linked with apolyfunctional compound, hence, a poly (acrylic acid), polyacrylate orreticulated acrylic acid and it's derivatives.

This makes up the hydration phase. After 24 hours a change in the colorof the distilled water will be observed from colorless to whitish and aslight increase in its viscosity.

In phase two, an industrial mixer with a capacity greater than 4 litersis used. The mixture obtained from the hydration phase of the solutes ispoured. Then, 650 mL of absolute alcohol (100%) is added. Thecombination is mixed for ten minutes observing the vortex of the mixturewhich will be inversely proportional to the viscosity. After ten minutesof mixing, 10 mL of glycerin, 0.5 mL of colorant and 5 mL of fragranceare added. The alcoholic degree is measured, to be confirmed it iswithin the range of 90 to 100% before proceeding with the next phase.

The third phase is neutralization. 1 mL of triethanolamine is addedwhile continuing to agitate the mixture. A change in coloration will beobserved again that depending on whether the vegetable coloring is used.Transparent blue should be observed which indicates that the properlygelled solution. Its viscosity is then verified to be within the rangeof between 2000 to 2500 centipoints.

Another Example of Ingredients of the Preservation Liquid/Gel

The following describes an example of manufacturing the preservationgel/liquid in a batch with these components:

1. DISTILLED WATER 3508 mL

2. CARBAPOL™ (a standardized polymer of acrylic acid cross-linked with apolyfunctional compound) 160 grams

3. TRIETHYLENE GLYCOL 80 grams

4. NIPASOL™ NIPAGIN™ (a standardized water soluble short-chain paraben)0.4 grams.

5. TRIETHANOLAMINE 0.33 Grams

6. VEGETABLE COLORING 1.5 cc

7. ESSENTIAL AROMATICS 30 cc

8. 70% ISOPROPYL ALCOHOL 1000 mL

The preparation process of this variation of the formula is similar tothat disclosed in the other examples described above.

An important version of the process can be fairly described as a processfor making a tissue preservation gel resulting from the ration resultingfrom the following components and comprised of the steps: combining andmixing in a vessel 350 mL distilled water, 5 g of a reticulated acrylicacid, or similar gelling agent and 1 g of a water soluble short-chainparaben. Then, sealing the vessel and waiting at least 24 hours forthese components to combine. Then, adding to the vessel 650 mL isopropylalcohol and mixing together in the vessel for at least 10 minutes. Then,adding and mixing to the vessel 10 mL glycerin. Then, measuring thealcoholic degree of the contents of the vessel and proceeding only whenthe alcoholic degree is within 90% to 100% or making adjustments to themixture to ensure it is in this range. Then, adding by mixing into thevessel 1 mL total of at least one of triethanolamine or propyleneglycol; measuring a viscosity of the contents of the vessel. Then,determining that the tissue preservation gel is correct only if theviscosity is between 2000 to 2500 centipoints, and if not makeadjustments to ensure this viscosity. Optionally, the process may becharacterized in that the water soluble short-chain paraben is Nipagin™(a standardized water soluble short-chain paraben). Optionally, thereticulated acrylic acid is Carbapol™ (a standardized polymer of acrylicacid cross-linked with a polyfunctional compound). Optionally, with theglycerin is included about 0.5 mL colorant. Optionally, with theglycerin is included about 5 mL fragrance, for example an essential oil.This formula includes it use that is fairly described as a process forpreserving a tissue comprised of the steps: submersing for at least 24hours the tissue to be preserved in a vessel containing a solution ofabout 0.9% hypertonic sodium chloride where a volume of the solution isat least 5 times, to about 10 times greater than a volume of the tissue.Then, replacing the gel with a first concentration of isopropyl alcoholand submerging the tissue. Then, replacing the first concentration ofisopropyl alcohol with a second greater concentration of isopropylalcohol and submersing the tissue. Then, retaining the tissue in thesecond greater concentration of isopropyl alcohol until the tissueachieves a paraffin stage. Then, submersing the tissue in a vesselcontaining the gel in claim 1.

The foregoing description conveys the best understanding of theobjectives and advantages of the present invention. Differentembodiments may be made of the inventive concept of this invention. Itis to be understood that all matter disclosed herein is to beinterpreted merely as illustrative, and not in a limiting sense.

I claim:
 1. A process for making a tissue preservation gel in theresulting ratio comprised of the steps: a) combining and mixing in avessel 350 mL distilled water, 5 g of a reticulated acrylic acid and 1 gof a water soluble short-chain paraben; b) sealing the vessel andwaiting at least 24 hours; c) adding to the vessel 650 mL isopropylalcohol with an alcoholic degree between 90% to 100%; d) mixing thevessel for at least 10 minutes; e) adding and mixing to the vessel 10 mLglycerin; f) adding and mixing into the vessel 1 mL total of at leastone of triethanolamine or propylene glycol; g) measuring a viscosity ofthe contents of the vessel; and h) selecting the contents of the vesselas the tissue preservation gel with the viscosity between 2000 to 2500centipoints.
 2. The process of claim 1 further characterized in thatwith the glycerin is included an additional 0.5 mL colorant.
 3. Theprocess of claim 1 further characterized in that with the glycerin isincluded an additional 5 mL fragrance.
 4. A process for making a tissuepreservation gel in the resulting ratio comprised of the steps: a)combining and mixing in a vessel 350 mL distilled water, 5 g of areticulated acrylic acid and 1 g of a water soluble short-chain paraben;b) sealing the vessel and waiting at least 24 hours; c) adding to thevessel 650 mL isopropyl alcohol with an alcoholic degree between 90% to100%; d) mixing the vessel for at least 10 minutes; e) adding and mixingto the vessel 10 mL glycerin, 5 mL fragrance and 0.5 mL colorant; f)adding and mixing into the vessel 1 mL total of at least one oftriethanolamine or propylene glycol; g) measuring a viscosity of thecontents of the vessel; and h) selecting the contents of the vessel asthe tissue preservation gel with the viscosity between 2000 to 2500centipoints.
 5. A method of making a tissue preservation gel for theconservation of biological specimens comprised of the following steps:for a proportionate ratio totaling of 2273.05 mL a) combining and mixingin a vessel: 1250 mL distilled water and at least 50 g of reticulatedacrylic acid and at least 0.2 g of a water soluble short-chain paraben;b) sealing the vessel and waiting at least 24 hours; c) adding to thevessel at least 1000 mL of a miscible antiseptic, selected from thegroup consisting of isopropyl alcohol, ethanol with an alcoholic degreebetween 90% to 100%; d) mixing the vessel for at least 10 minutes; e)adding to the vessel and mixing at least 10 mL of a low molecular weightpolyol selected from: glycerin, polyethylene glycol (PEG) andpolypropylene glycol (PPG); f) adding and mixing into the vessel atleast 1 mL total of a surfactant selected from: triethanolamine (TEA) ordimethicone; g) adjusting the viscosity of the mixture to a specificweight of neutral buoyancy and a viscosity between 2000 to 2500centipoints.
 6. The method of claim 5 further characterized in that thesuitable water soluble short-chain paraben is selected from any of:methyl paraben or propylparaben.
 7. The method of claim 5 furthercharacterized in that the reticulated acrylic acid is selected from anyof: acrylic acid or carbomer.
 8. The method of claim 5 furthercharacterized in that with the low molecular weight polyol includes atleast 0.5 mL of a colorant.
 9. The method of claim 5 furthercharacterized in that with the low molecular weight polyol includes atleast 5 mL of a fragrance.